TWI396741B - Vero cell-based influenza virus strains and vaccines - Google Patents

Description

VERO CELL-BASED INFLUENZA VIRUS STRAINS AND VACCINES

The present invention relates to influenza virus strains, in particular to influenza A virus strains suitable for increasing the production of influenza A vaccine in mammalian host cells, and vaccines produced.

Influenza viruses, particularly influenza A and B viruses, are associated with major outbreaks of influenza and cause severe rickets and mortality in animals. Infected type B is usually milder than type A. Influenza A viruses usually carry the original birds, but may infect several mammals. The subtypes of the currently known influenza A virus are all occurring in regional birds. Birds have the largest number and range of influenza strains. These influenza strains continue to experience micro-variation of antigens, perhaps driven by the pressure of selective antibodies produced by humans, resulting in seasonal epidemics in some areas. Recently, the highly pathogenic H5N1 subtype of avian influenza A virus has been found to spread throughout East Asia. Some human H5N1 strains have been isolated, and in 2005, attenuated H5N1 vaccine strains with hemoglobin-deficient multi-salt base were produced by reverse transcription engineering (Emerging Infectious Disease; Vol.11, No.10). , October 2005, 1515-1521.)

The influenza virus is an RNA virus belonging to the Orthomyxoviridae family, including the genome of negative-negative RNA, which can be encoded as approximately 10 influenza proteins. These 10 influenza proteins contain RNA-mediated RNA polymerase proteins (PB2, PB1 and PA), nuclear proteins (NP), neuraminidase (NA), and hemagglutinin proteins (HA, which are formed by enzyme digestion to form HA1). Combined with HA2 subunits), matrix proteins (M1 and M2) and non-structural proteins (NS1 and NS2) (Krug et al., In The Influenza Viruses, RM Krug, ed., Plenum Press, New York, 1989, pp. 89-152).

In the past few decades, fertilized eggs have been the main host system for breeding influenza viruses to produce vaccines. In addition, African green monkey kidney cells (Vero cell) and Madin-Darby's dog kidney epithelial cells (MDCK) have been widely used in the manufacture of seasonal influenza vaccines. (Audrey et al., Expert Opin Biol Ther 4(5): 709-17 (2004); Ghendon et al., Vaccine 23(38): 4678-84 (2005)).

NIBRG-14 is the current influenza H5N1 vaccine strain, provided by the National Institute of Biology and Serology Control (NIBSC), a HA and NA gene containing A/Vietnam/1194/2004 (H5N1) and A/ A reassortant virus made from the other six internal gene parts of PuertoRico/8/1934 (H1N1). This vaccine strain was found to grow efficiently in chicken embryonic eggs and MDCK cells, but not in African green monkey kidney cells. (Govorkova, E.A., S. Kodihalli, et al., Dev Biol Stand 98: 39-51 (1999)). However, in the case of a worldwide influenza pandemic, the use of eggs to make influenza vaccines will not be sufficient, while MDCK cells have worrying potential carcinogenicity.

Therefore, there is a need for a strain of influenza virus that can grow rapidly in African green monkey kidney cells to increase the production of influenza vaccine. The present invention satisfies such needs.

In one aspect, an embodiment of the invention relates to a strain of influenza A virus that is efficiently grown in a mammalian host cell and suitable for increasing production in a mammalian host cell, the influenza A strain comprising the following group selected At least one gene that modifies the influenza protein: (a) a modified influenza polymerase protein (PB1) comprising an leucine group at position 195 relative to SEQ ID: 1; (b) a modified flu The polymerase protein (PB2), including a tyrosine group located at position 359 relative to SEQ ID NO: 3; (c) a modified influenza nucleoprotein (NP), including positions relative to the sequence identifier: 5 One aspartic acid and two phenylalanine groups at positions 255, 410 and 446; (d) a modified influenza interstitial protein (M), including amphetamine at position 236 relative to SEQ ID NO: 7 Acidic group; (e) a modified influenza polymerase protein (PA), including position relative to Column identification number: 9 histidine or glycine acid group at position 465 or 494; and (f) a modified influenza non-structural protein (NS), including (i) position relative to sequence identification number: 11 of 90 And two prolyl groups at position 110; or (ii) a stop codon at 122 relative to SEQ ID NO: 11; wherein expression of the at least one gene results in a strain of influenza A in a mammalian host cell The output increased.

In another aspect, embodiments of the invention are related to influenza vaccines. The vaccine includes an influenza A virus strain according to an embodiment of the present invention and a pharmaceutically acceptable carrier. Embodiments of the invention are also related to methods of preparing influenza vaccines. This method comprises the combination of an influenza virus strain and a pharmaceutically acceptable carrier according to an embodiment of the invention.

In another aspect, an embodiment of the invention is directed to an isolated nucleic acid molecule encoded as at least one modified influenza protein selected from the group consisting of: (a) a modified influenza polymerase protein (PB1), comprising a modified flu polymerase protein (PB2) relative to sequence identification number: 1 at position 195; (b) a modified influenza polymerase protein (PB2) comprising a tyrosine group at position 359 relative to SEQ ID NO:3; (c) a modified influenza nucleoprotein (NP) comprising an aspartic acid and two phenylalanine groups at positions 255, 410 and 446, respectively, relative to SEQ ID: 5; (d) a modified flu The interstitial protein (M) includes a phenylalanine group at position 236 relative to SEQ ID NO: 7; (e) a modified influenza polymerase protein (PA), including a position relative to the sequence identifier: a histidine or glycine group at position 465 or 494; and (f) a modified influenza non-structural protein (NS) comprising (i) a position at positions 90 and 110 relative to the sequence identifier: 11 a proline group; or (ii) a stop code at 122 relative to the sequence identifier: 11; Wherein the expression of the at least one gene results in increased production of the influenza A virus strain in the mammalian host cell.

An embodiment of the invention is directed to the above-described isolated modified influenza protein.

Other features and advantages of the invention will be apparent from the description. The detailed description of the invention and the preferred embodiments and additional patent application scope are included.

The present invention relates to highly grown influenza virus strains suitable for replication in mammalian host cells. According to an embodiment of the present invention, the vaccine strain of H5N1 is easily subcultured in mammalian primary cells such as African green monkey kidney cells, such as NIBRG-14, which has been selected, isolated and characterized.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as the ordinary skill to which the invention pertains, and the specific terms used in this application are used frequently, and the meaning should be given in the foregoing. It must be noted that the singular forms "a", "an" or "the"

Herein, a reassortant virus refers to a portion of a gene that is derived from two or more different virus strains at the same time. The recombinant virus will also have the characteristics of the parental lineage. For example, a recombinant virus can obtain a portion of a gene encoded by an antigenic protein (such as the HA and NA genes) from a strain of interest, and encode a gene portion of a virus (or an attenuated virus) suitable for growth into other viral proteins, such as Polymerase complexes (PB2, PB1 and PA genes) and other viral proteins (such as non-glycoprotein genes, including M genes, S genes and NP genes). Thus, the recombinant virus itself carries the desired antigenic properties or a provenance strain that is efficiently produced in the host cell. Such a recombinant virus is a "viral seed" that is expected to produce a virus required for preparing a vaccine. In one embodiment of the invention, the recombinant virus comprises NIBRG-14 strain and any NIBRG-14 modified virus strain, such as vero-15 strain and vero-16 strain.

As used herein, the term "modified strain" means a strain of a virus obtained by successive passage of a first generation virus strain and/or via a first generation virus in a mammalian host cell and its progeny. In one embodiment of the invention, the modified strain is more than the first generation disease The strain has increased the production of the virus in mammalian host cells.

As used herein, the term "influenza protein" means any polypeptide or protein encoded by an influenza gene.

As used herein, a "gene" refers to a portion of a nucleic acid molecule that produces a peptide, polypeptide or protein, and a messenger RNA (mRNA) is encoded into these protein species, including a cryptographic region, a 5' non-translated region preceding the non-cryptographic region ("5 'UTR' followed by the 3' non-translated area ("3' UTR" cipher area. The "gene" also contains non-cryptins (interns) interspersed in two separate cryptodomain sequences (exonons). The gene may further comprise regulatory sequences that control the expression of the gene. Regulatory sequences include promoters, enhancers, and other elements that control expression, such as polyadenylation signals, ribosome binding (bacterial expression), and/or an operator. Promoter means a regulatory sequence involved in the binding of RNA polymerase to initiate transcription of a gene. The promoter is usually located upstream ("5' to) end of the transcription start position of the gene. The enhancer means a regulatory sequence that regulates gene expression according to the form of the distally determined orientation. The cryptodomain refers to the coding for amino acid. This part of the gene with the start and stop signals of the corresponding three sets of polypeptide translation.

As used herein, an "influenza gene" can be any gene originally isolated and/or identified by any influenza virus. In a particular embodiment of the invention, the influenza gene is a gene originally isolated and/or identified by an influenza A virus.

As used herein, the term "modified influenza protein" means a protein comprising one or more modifications in an amino acid sequence that has not been modified or referenced to an influenza protein. Modifications include, for example, insertions, substitutions, or deletions of one or more amino acid groups located in an unmodified or reference influenza protein sequence. Thus, the modified influenza protein can have at least one modified amino acid group and/or a code occupying a position that has not been modified or referenced by the reference influenza protein.

For simplicity and clarity, modification of the modification in influenza proteins is identified by the relative position of the influenza protein in its particular reference influenza strain, such as influenza NIBRG-14. However, this identification does not limit the modified influenza protein to the modified influenza NIBRG-14 protein. Modified influenza protein can be any influenza virus The influenza protein is modified to contain a modification relative to the position of the NIBRG-14 protein.

As used herein, the term "amino acid substitution" refers to the presence of an amino acid in an identified position in the amino acid sequence of a modified protein. Amino acid substitutions occur in relation to any other amino acid that may occupy the unmodified or reference protein amino acid position. Modified proteins consisting of amino acid substitutions may also contain other modifications.

Herein, the modified influenza polymerase protein (PB1), including the leucine group at 195 relative to SEQ ID NO: 1, means to modify the influenza polymerase protein (PB1), relative to the sequence identifier: There is an leucine group at 195 of 1. The influenza polymerase protein (PB1) is modified, not limited to the modified polymerase protein (PB1) from influenza NIBRG-14, and the lysine is substituted with leucine at 195 relative to SEQ ID NO: 1. The leucine group is present relative to any other amino acid group that may occupy the unmodified or reference polymerase protein (PB1). The modified influenza polymerase protein (PB1) may be a modified polymerase protein (PB1) in influenza A virus other than NIBRG-14, as long as it is a modified polymerase protein (PB1) relative to the sequence identifier: There are leucine groups at 195. The modified influenza polymerase protein (PB1) also contains modifications other than the specified leucine group. As used herein, "SEQ ID NO: 1 position 195" means that the amino acid group is the 195th amino acid from the N-terminus of the sequence identifier: 1. From the current description, the position of the polymerase protein (PB1) from other influenza A viruses in sequence identification number: 1 relative to position 195 can be identified without difficulty by known techniques, such as sequence alignment analysis. Similar instructions apply to other modified influenza proteins, such as PB2, NP, M, PA and NS, which are described herein in a similar manner.

As used herein, the term "modified influenza polymerase protein (PB1)," means a protein modified by one or more of the amino acid sequences of an unmodified or reference influenza polymerase protein (PB1). The influenza polymerase protein (PB1) is a monomer encoded by the influenza PB1 gene and is an influenza RNA-dependent RNA polymerase. The influenza PB1 gene falls on the negative strand portion 2 of the influenza A genital part. An example containing the influenza PB1 gene but not the PB1 gene of influenza A virus, for example, the sequence number isolated from the NIBRG-14 virus: 2, from influenza A/Hong Kong/1073/99 (H9N2) isolated nucleic acid library accession number NC_004911.1, nucleic acid library accession number NC_007372.1 isolated from influenza A/New York/392/2004 (H3N2), from influenza A/Korea /426/68 (H2N2) isolated nucleic acid library accession number NC_007375.1, nucleic acid library accession number NC_002021.1 isolated from influenza A/Puerto Rico/8/34 (H1N1), from influenza A/Goose/ The nucleic acid database registration number NC_007358.1 isolated from Guangdong/1/96 (H5N1).

As used herein, the term "modified influenza polymerase protein (PB2)," means a protein that is modified at one or more of the amino acid sequences that have not been modified or referenced to the influenza polymerase protein (PB2). The influenza polymerase protein (PB2) is a monomer encoded by the influenza PB2 gene and is an influenza RNA-dependent RNA polymerase. The influenza PB2 gene falls on the negative strand fragment 1 of the influenza A gene body segment. Examples containing the influenza PB2 gene are not restricted to the PB2 gene of influenza A virus, for example, the sequence number isolated from the NIBRG-I4 virus: 4, isolated from influenza A/Hong Kong/1073/99 (H9N2) Nucleic Acid Library Registry Number NC_004910.1, Nucleic Acid Library Accession Number NC_007373.1 isolated from influenza A/New York/392/2004 (H3N2), isolated from influenza A/Korea/426/68 (H2N2) Nucleic acid library accession number NC_007378.1, nucleic acid library accession number NC_002023.1 isolated from influenza A/Puerto Rico/8/34 (H1N1), or isolated from influenza A/Goose/Guangdong/1/96 (H5N1) The nucleic acid database registration number NC_007357.1.

As used herein, the term "modified influenza nucleoprotein (NP)," means a protein modified by one or more of the amino acid sequences that have not been modified or referenced to influenza nucleoprotein (NP). Influenza nuclear protein (NP), encoded by the influenza NP gene, is an influenza nucleoprotein. The influenza NP gene falls on the negative strand fragment 5 of the influenza A genome. Examples containing the influenza NP gene are not restricted to the NP gene of influenza A virus, for example, the sequence number isolated from the NIBRG-14 virus: 6, from the influenza A/Hong Kong/1073/99 (H9N2) Isolation of Nucleic Acid Library Registry Number NC_004905.2, Nucleic Acid Library Accession Number NC_007369.1 isolated from influenza A/New York/392/2004 (H3N2), isolated from influenza A/Korea/426/68 (H2N2) Gene bank identification number: 3655111, nucleic acid library accession number NC_002019.1 isolated from influenza A/Puerto Rico/8/34 (H1N1), or isolated from influenza A/Goose/Guangdong/1/96 (H5N1) The gene bank registration number NC_007360.1.

As used herein, the term "modified influenza interstitial protein (M)," means a protein modified by one or more of the amino acid sequences comprising an unmodified or reference influenza interstitial protein (M). Influenza interstitial protein (M), which is the larger of the two matrix proteins, is encoded by the influenza M gene. The influenza M gene falls on the negative strand fragment 7 of the influenza A gene segment. An example containing the influenza M gene but not limited to the M gene of influenza A virus, for example, the sequence identifier number isolated from the NIBRG-I4 virus: 8, isolated from influenza A/Hong Kong/1073/99 (H9N2) Nucleic Acid Library Registry Number NC 004907.1, Nucleic Acid Library Registry Number isolated from influenza A/New York/392/2004 (H3N2). NC 007367.1, Nucleic acid library registration number isolated from influenza A/Korea/426/68 (H2N2). NC_007377.1, nucleic acid library accession number isolated from influenza A/Puerto Rico/8/34 (H1N1). NC_002016.1, or nucleic acid library accession number NC_007363.1 isolated from influenza A/Goose/Guangdong/1/96 (H5N1).

As used herein, the term "modified influenza polymerase protein (PA)," means a protein modified by one or more of the amino acid sequences comprising an unmodified or reference influenza polymerase protein (PA). Influenza polymerase protein (PA), encoded by the influenza PA gene, is an influenza polymerase PA. The influenza PA gene falls on the negative strand fragment 3 of the influenza A gene segment. Examples containing the influenza PA gene are not restricted to the PA gene of influenza A virus, for example, the sequence number of the isolate from the NIBRG-I4 virus: 10, isolated from influenza A/Hong Kong/1073/99 (H9N2) Nucleic Acid Library Registry Number NC_004912.1, from influenza A/New York/392/2004 (H3N2) The isolated nucleic acid library registration number NC_007371.1, the nucleic acid library registration number NC_007376.1 isolated from the influenza A/Korea/426/68 (H2N2), separated from the influenza A/Puerto Rico/8/34 (H1N1) The nucleic acid library registration number NC_002022.1, or the nucleic acid library registration number NC_007359.1 isolated from influenza A/Goose/Guangdong/1/96 (H5N1).

As used herein, the term "modified influenza non-structural protein (NS)," means a protein modified by one or more of the amino acid sequences that have not been modified or referenced to the influenza non-structural protein (NS). Influenza non-structural protein (NS), encoded by the influenza NS gene, is an influenza non-structural protein. The influenza NS gene falls on the negative strand segment 8 of the influenza A gene fragment. An example of an influenza NS gene, but not limited to the NS gene of influenza A virus, for example: SEQ ID NO: 12, isolated from NIBRG-I4 virus, isolated from influenza A/Hong Kong/1073/99 (H9N2) Nucleic Acid Library Registry Number NC_004906.1, Nucleic Acid Library Accession Number NC_007370.1 isolated from influenza A/New York/392/2004 (H3N2), isolated from influenza A/Korea/426/68 (H2N2) Gene bank identification number 3655110, nucleic acid library accession number NC_002020.1 isolated from influenza A/Puerto Rico/8/34 (H1N1), nucleic acid isolated from influenza A/Goose/Guangdong/1/96 (H5N1) Database registration number NC_007364.1.

As used herein, the term "nucleotide sequence" means a combination of deoxyribonucleic acid or ribonucleic acid that is polymerized to form a single or double strand. The nucleic acid sequence may be composed of the following natural nucleotide bases and/or analogs of synthetic natural nucleotides: thymine, adenine, cytosine, guanine, and uracil; abbreviated as T, A, C, respectively. G, and U.

As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule that is substantially separated from at least one of the nucleic acids of natural origin, or substantially frees at least one chemical precursor when the nucleic acid molecule is chemically synthesized or Other compounds. "Isolated" nucleic acid molecules can also be derived from the genome of the organism However, among the nucleic acid molecules at the 5' and 3' ends of the DNA, the nucleic acid of at least one nucleic acid sequence is substantially liberated. A nucleic acid molecule that is "substantially derived from" or "substantially free from" other nucleic acid molecules or compounds used to prepare the nucleic acid molecule should be less than about 30%, 20%, 10%, 5%, and less, and in other nucleic acid molecules. Preferably, less than 1% (dry weight) of the compound (see herein "Contaminated Nucleic Acid Molecules" or "Contaminated Compounds").

Isolated nucleic acid molecules include, but are not limited to, nucleic acid molecule classifications independent of other sequences (eg, cDNA or gene fragments produced by PCR and restriction enzymes), and nucleic acid molecules embedded in self-replicating plastid vectors, viruses (eg, retrovirus, adenovirus or eruption virus), or embedded in the genome of prokaryotic or eukaryotic cells. In addition, the isolated nucleic acid molecule can comprise a hybridized or fused portion. The isolated nucleic acid molecule can be a nucleic acid sequence: (i) in vitro assay amplification, such as polymerase chain reaction (PCR) (ii) by synthetic means, such as chemical synthesis; (iii) recombination by plasmid or (iv) fragmentation Purified by electrophoresis or layer chromatography.

As used herein, the terms "polypeptide" and "protein" are used interchangeably herein to mean an amino acid chain in which the amino acid residue is linked by a peptide bond or a modified biopeptide bond. The amino acid chain can be adapted to any length greater than two amino acids. The terms "polypeptide" and "protein" also encompass a variety of modified styles unless otherwise noted. This modified style may be a natural or chemically modified style. Modifications include, but are not limited to, the following examples of glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, and the like. Modification also includes intramolecular cross-linking and a portion of covalent linkages with a wide variety of moieties, such as lipids, flavin, biotin, polyethylene glycol or related derivatives. In addition, modification may also include cyclizatioh, branching and cross-linking. Amino acids other than the twenty essential amino acids are encoded by the codons of the genes and are also included in the polypeptide.

"Isolated protein" and "isolated polypeptide" are essentially natural A protein isolated from at least one of the source proteins, or a compound that is substantially freed by at least one chemical step or chemically synthesized by the protein. The protein is substantially "isolated" or less than about 30%, 20%, 10%, or 5% and less by free protein or other chemically prepared protein, and preferably less than other proteins or compounds. 1%, (dry weight) (see "Contaminated Proteins" or "Contaminated Compounds")

Isolated proteins can be in several different natural forms. The isolated protein may be present in a full-length nascent or unprocessed polypeptide or a partially processed polypeptide or a processed polypeptide binding format. Full-length nascent polypeptides can be post-translationally modified by specific protein cleavage programs, resulting in the formation of full-length nascent polypeptide fragments. Fragments or physically related fragments have biological activities associated with full length nascent polypeptides, however the size of individual fragment activity will vary.

An isolated polypeptide or an isolated protein can be a non-naturally occurring polypeptide. For example, an isolated polypeptide can be a "hybrid polypeptide." It may also be derived from the addition or deletion or substitution of a natural polypeptide via an amino acid. An isolated polypeptide may also be a "purified polypeptide" as used herein to refer to a particular polypeptide, substantially homologously prepared, substantially free of other cellular constituents, other polypeptides, viral material or culture medium, or when the polypeptide is chemically synthesized, chemically Precursor or an accessory associated with chemical synthesis. "Purified polypeptides" can be obtained from natural or recombinant host cells using standard purification techniques, or by chemical synthesis, as will be apparent to those skilled in the art.

"Isolated influenza virus strain" or "isolated influenza virus" is basically a virus isolated from at least one other virus in the natural environment, or when the virus is derived or modified by the parental virus strain to be substantially free. In addition, it is derived from the parent strain. The virus is "substantially separated" or "substantially free" from viruses or other viruses less than about 30%, 20%, 10%, or 5% and less, and other viruses are preferably less than 1% (dry) Heavy) (see "Contaminated Viruses" in this article).

In one aspect, the invention is therefore directed to an isolated influenza strain that can produce high titers in mammalian cells. Embodiment of the present invention relates to isolated influenza The viral strain comprises at least one of the at least one modified influenza protein, and the performance of the at least one modified protein results in an increased production of influenza virus. According to an example of the invention, the host cell may be selected from a combination of mammalian cells suitable for the preparation of a human vaccine. In a particular example of the invention, the mammalian cells may be African green monkey kidney cells prepared according to World Human Tissue (WHO) approval and certified preparation of vaccines. At least one modified influenza protein selected by the following group: (a) a modified influenza polymerase protein (PB1) comprising an leucine group at position 195 relative to SEQ ID NO: 1; (b) A modified influenza polymerase protein (PB2) comprising a tyrosine group located at position 359 relative to SEQ ID NO: 3; (c) a modified influenza nucleoprotein (NP), including positional relative to sequence recognition No.: an aspartic acid and two phenylalanine groups at positions 255, 410 and 446; (d) a modified influenza interstitial protein (M), including position 236 relative to the sequence identifier: 7 a phenylalanine group; (e) a modified influenza polymerase protein (PA) comprising a histidine or glycine group at position 465 or 494 relative to SEQ ID NO: 9; and (f) Modification of influenza non-structural proteins (NS), including (i) two proline groups at positions 90 and 110 relative to SEQ ID NO: 11; or (ii) 122 relative to SEQ ID NO: 11 A stop code at the site; a particular example of an embodiment of the invention relates to the influenza virus strain Vero-15. The isolated and modified influenza virus strain Vero-15 includes a protein encoded by the modified influenza PB1 gene, including the amino acid sequence of SEQ ID NO: 1, substituted at 195 of SEQ ID NO: 1 for the leucine group. A protein encoded by the modified influenza PB2 gene includes the amino acid sequence of SEQ ID NO: 3, substituted at 359 of SEQ ID NO: 3 to a tyrosine group. A protein encoded by the modified influenza PA gene includes the amino acid sequence of SEQ ID NO: 9 and is substituted with a glycine group at 494 of SEQ ID NO: 9. Modified influenza NS gene The encoded protein includes the amino acid sequence of SEQ ID NO: 11 and is substituted with two proline groups at 90 and 110 of SEQ ID NO: 11. The influenza virus strain Vero-15 further comprises a protein encoded by the HA gene comprising an amino acid sequence of SEQ ID NO: 13 comprising a protein encoded by the NA gene comprising an amino acid sequence of SEQ ID NO: 15 encoding the NP gene. The protein includes the amino acid sequence of SEQ ID NO: 5, and the protein encoded by the M gene includes the amino acid sequence of SEQ ID NO: 7.

From a nucleotide point of view, the influenza virus strain Vero-15 includes the PB1 gene having the nucleotide sequence of SEQ ID NO: 2, and is substituted with thymidine at 583, 584 and 1257 of SEQ ID NO: 2 The PB2 gene having a nucleotide sequence of SEQ ID NO: 4 was substituted with adenine in 1737, substituted with trithymidine at 1077 of SEQ ID NO: 4, and a PA having a nucleotide sequence of SEQ ID NO: 10. The gene was substituted with guanine at 1482 of SEQ ID NO: 10, and the NS gene having the nucleotide sequence of SEQ ID NO: 12 was substituted with cytosine at positions 271, 331 and 335 of SEQ ID NO: 12. The influenza virus strain Vero-15 includes an HA gene having a nucleotide sequence of SEQ ID NO: 14, and an NA gene having a nucleotide sequence of SEQ ID NO: 16. The influenza virus strain Vero-15 further includes an NP gene having a nucleotide sequence of SEQ ID NO: 6, and an M gene having a nucleotide sequence of SEQ ID NO: 8.

Comparing the nucleotide sequences of Vero-15 and NIBGR-14, there are 1 to 4 different nucleotides in the PB2, PB1, PA and NS parts, but the nucleotide sequences of HA, NA, NP and M fragments are NIBGR-14 is no different. A detailed comparison is shown in Table 1 and Figures 5 through 10.

According to another example of an embodiment of the invention, a modified influenza virus strain Vero-16 is provided. The influenza virus strain Vero-16 includes a protein encoded by a modified influenza PB1 gene, and has an amino acid sequence of SEQ ID NO: 1, which is substituted with leucine group at 195 of SEQ ID NO: 1. A modified protein encoding the influenza PB2 gene, having the amino acid sequence of SEQ ID NO: 3, substituted at 359 of SEQ ID NO: 3 to a tyrosine group. a modified influenza NP gene encoded into The protein, having the amino acid sequence sequence of SEQ ID NO: 5, was substituted with an aspartic acid and two phenylalanine groups at 255, 410 and 446 of SEQ ID NO: 5, respectively. A protein encoded by the modified influenza M gene, having the amino acid sequence of SEQ ID NO: 7 substituted at 236 of SEQ ID NO: 7 to a phenylalanine group. A protein encoded by the modified influenza PA gene, having the amino acid sequence of SEQ ID NO: 9 substituted at 465 of SEQ ID NO: 9 to a histidine group. A protein encoded by the modified influenza NS gene having the amino acid sequence of SEQ ID NO: 11 and a substitution at 122 of SEQ ID NO: 11 is a stop code. The influenza virus strain further includes a protein encoded by the HA gene, and has an amino acid sequence of SEQ ID NO: 13, including a protein encoded by the NA gene, and has an amino acid sequence of SEQ ID NO: 15.

From a nucleotide point of view, the influenza virus strain Vero-16 includes the PB1 gene having the nucleotide sequence of SEQ ID NO: 2, and is substituted with three thymines at 583, 584 and 1257 of SEQ ID NO: 2 And 1737 is substituted with adenine, and the PB2 gene having the nucleotide sequence of SEQ ID NO: 4 is substituted with adenine at 1077 of SEQ ID NO: 4, and has a nucleotide sequence of SEQ ID NO: 6. The NP gene was substituted with four thymines at 921, 1229, 1337 and 1413, respectively, in sequence identification number: 6, and the M gene having the nucleotide sequence of SEQ ID NO: 8 was found at 694 of sequence number: 8 and The 767 gene was substituted with two thymidines, and the PA gene having the nucleotide sequence of sequence number: 10 was substituted with three cytosines at 895, 1054 and 1394 of SEQ ID NO: 2, with sequence identification number: The NS gene of the nucleotide sequence of 12 deletes the nucleotide at 365 of SEQ ID NO: 2. The influenza virus strain Vero-16 includes an HA gene having a nucleotide sequence of SEQ ID NO: 14 and an NA gene having a nucleotide sequence of SEQ ID NO: 16.

Comparing the nucleotide sequences of Vero-16 and NIBGR-14, there are 1 to 5 different nucleotides in the PB2, PB1, PA, NS, NP and M parts, but the HA and NA partial nucleotide sequences It is no different from NIBGR-14. A detailed comparison is shown in Table 1 and Figures 5 through 10.

According to the present invention, there is provided a method of improving the production of a H5N1 influenza vaccine. This An improved method involves the use of a modified seed virus. The modified seed virus comprises at least one modification selected from the group consisting of: a protein encoded by the modified influenza PB1 gene, comprising an leucine group at 195 relative to SEQ ID NO: 1, and a modified influenza PB2 gene encoding The resulting protein, comprising a tyrosine group at 359 relative to SEQ ID NO: 3, a protein encoded by a modified influenza NP gene, including a day at 255, 410 and 446 relative to SEQ ID NO: 5 A towline group and two phenylalanine groups, a protein encoded by the modified influenza M gene, comprising a phenylalanine group at 236 relative to SEQ ID NO: 7, and a protein encoded by the modified influenza PA gene, including A protein encoded by a modified NS gene relative to the 465 or 494 of the sequence identifier: 9 having a histidine group or a glycine group, including 2 at 90 and 110 relative to the sequence identifier: 11 The prolyl group has a stop code at 122.

From a nucleotide point of view, the modified seed influenza virus strain used to produce the H5N1 influenza vaccine comprises at least one modification selected from the group consisting of 583, 584 and 1257 of the modified PB1 gene at sequence identification number: 2, respectively. There are three thymines, and there is adenine at 1737. The modified PB2 gene has adenine at 1077 of sequence ID: 4, and the modified NP gene has guanine at 763 of sequence number: 6 and is at 921. There are four thymines at 1229, 1337 and 1413, and the modified M gene has two thymines at 694 and 706 of sequence identification number: 8, respectively, and the modified PA gene has guanine at 1482 of sequence identification number: 10 or There are three cytosines at 895, 1054 and 1394, and the modified NS gene has three cytosines at positions 271, 331 and 335 of SEQ ID NO: 12 or nucleotides at 365, respectively.

Other aspects of the invention are that the influenza vaccine according to an embodiment of the invention comprises at least one modified influenza strain, or any combination of the invention. The influenza virus strains according to the present invention may be attenuated or detoxified, or formulated and vaccinated, according to known methods as a vaccine for inducing an immune response in mammals. The antigenicity is determined by any standard method known in the art. The vaccine composition may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means that the molecular entity and composition are pharmaceutical tolerable and When given to humans, it does not induce typical allergies or similar unfortunate reactions. Such as gastrointestinal discomfort, dizziness and the like. The term "carrier" means that diluents, adjuvants, physiological saline and dextrose and glycerin are often used as carriers, particularly for injectable solutions. The term "adjuvant" refers to a complex or mixture that can increase the immune response of an antigen.

The vaccine of the invention may be transdermally inoculated, blood-containing, transmucosal, such as oral, nasal, rectal or transdermal. The method of inoculation includes transfusion, such as intravenous injection but is not limited to transvascular, intraarterial, intramuscular, subcutaneous, acupoint, intraperitoneal, intravenous, and intracranial.

The facets of the present invention are also associated with the isolation of isolated nucleic acid molecules into modified influenza proteins and their production, isolation and method of use. For example, nucleic acid molecules isolated in this manner can be used in the construction of influenza viruses to make them more efficient in host cells. The present invention finds that when an influenza virus comprises one or more of the modified influenza proteins described herein, the virus grows more efficiently in the host cell. Thus, in order to obtain a rapidly growing strain of virus, the nucleic acid molecule encoded as a modified influenza protein can be combined with the viral gene of interest by means of recombination or other manipulation of the gene. Such genetic methods may be known to those skilled in the art. An isolated nucleic acid molecule can also be used to recombine the production of a modified influenza protein encoded by an isolated nucleic acid molecule.

The invention is further related to the isolated modified influenza protein and its associated production methods, isolation and use. The protein so isolated can be used to produce antibodies to this modified protein.

The invention will now be further described with reference to the following specific but non-limiting examples.

example

Modification of NIBRG-14 in African green monkey kidney cells

The influenza virus strain NIBRG-14 is supplied by the National Institute of Biological and Serological Control (NIBSC) and cultured in chicken embryo eggs. The African Green Monkey Kidney Cell approved by the World Human Tissue (WHO) vaccine was provided by the Taiwan Disease Control Bureau and was cultured using M199 medium (Gibco BRL, USA) containing 5% fetal bovine serum (FBS). The number of generations is between 135 and 150 generations. The influenza virus strain NIBRG-14 was subcultured in African T-cells in T75 containers for 3 consecutive times. During the culture, 2 μg/ml of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) trypsin (Sigma, St.Louis, MO). The virus suspension after subculture was collected and placed in a 96-well plate for colonization, followed by two colonizations in a 6-well plate, and 4 μg/ml TPCK-trypsin was added to the culture medium at the time of colonization. For each plant, the cumulative amount of virus was estimated by cytopathic effect and HA titration. HA titration was performed using 96-well plates using turkey red blood cells (RBC) using standard techniques. Viral infection titer is measured by 50% tissue culture infection dose analysis (TCID50) based on cytopathic effect or plaque assay based on plaque forming unit (PFU) (WHO is used in WHO) Method for diagnosis and monitoring of animal influenza, WHO 2002), 4 μg/ml of TPCK-trypsin was added to the culture medium during the test.

The influenza H5N1 vaccine strain obtained by colonization was subsequently infected with African green kidney monkey cells grown in a T25 container and 4 μg/ml of TPCK-trypsin was added to the medium, and the virus suspension on the third day after infection was collected for HA. Titration and viral infection titer testing. Two strains of influenza H5N1 vaccine strain that can grow rapidly and adapt to African green monkey kidney cells (Vero-adapted) were selected and named Vero-15 and Vero-16, respectively. The growth curve of NIBRG-14 in African green monkey kidney cells was measured using infection density (MOI) of 10-1, 10-2 and 10-3 TCID50/cells, respectively. The infection density (MOI) of domesticated African green monkey kidney cells (Vero-adapted) Vero-15 and Vero-16 was 10-3 and 10-4 TCID50/cells.

As shown in Figures 1 and 2, NIBRG-14 formed small, unidentified plaques 6 days after infection with African green monkey kidney cells, with a spike of virus titration of approximately 106 PFU/ml. Two high-growth plants (Vero 15 and Vero-16) were selected from large plaques by successive passage and plaque purification in African green monkey kidney cells. Referring to Figure 1, Figure 3 and Figure 4, the two strains modified by African green monkey kidney cells, Vero 15 and Vero-16 form clear and large plaques after 3 days of infection with African green monkey kidney cells. The peak of the virus titration is about 108 TCID50/ml. further These two strains of virus modified by African green monkey kidney cells were used to make a virus stock solution in a T75 or T150 container. The virus stocks of the two strains modified by African green monkey kidney cells have similar viral titers (Vero-15 is 1.14 x 108 PFU/ml and 1.81 x 108 TCID50/ml and Vero-16 is 0.99 x 108 PFU/ Ml and 2.96 x 108 TCID50/ml).

Identification of viral nucleotide sequences and alignment

The viral RNA of each strain was extracted using a commercial kit (Geneaid, Taoyuan, Taiwan). HA and NA in the extracted viral RNA were amplified using one-step RT-PCR (Promega, Madison, WI), while the other six gene fractions were subjected to two-step RT-PCR (two-step RT-). PCR) (Invitrogen, Rockville, MD). The single nucleotide primers used in this study are that the sequence is based on the gene sequence of NIBRG-14 without difficulty. The amplified DNA was sequenced using an ABI 3730 XL DNA Analyzer (Applied Biosystems Inc, Foster City CA).

Table 1 lists the number of differences obtained by comparing the Viro-15 and Vero-16 and NIBRG-14 nucleic acid sequences and amino acid sequences of domesticated African green monkey kidney cells (Vero-adapted). Note the Vero-15 strain, compared with NIBRG-14. Although the nucleic acid sequences of HA, NA, NP and M are not different from NIBRG-14, they differ from one to four nucleotides in four parts, as shown in Figure 1. Shown are PB2, PB1, NS and PA. Note the Vero-16 strain, compared with NIBRG-14. Although the nucleic acid sequences of HA and NA are not different from NIBRG-14, they differ from one to five nucleotides in six parts, as shown in Figure 1 for PB2. , PB I, PA, NS, NP and M.

* Vero-16 has a nucleotide to be deleted, resulting in a stop code at 122 of the sequence identification number: 11.

NT: nucleotide

AA: Amino acid

Sequence analysis of amino acid (AA) and nucleotide (NT) of three influenza strains is shown in Fig. 5 to 10.

Antigenicity test

To measure the antigenic relationship between NIBRG-14 and modified African green monkey kidney cell virus strains. Anti-NIBRG was measured using multiple sheep anti-NIBRG-14 standard antisera (NIBSC) using the standard hemagglutination inhibition (HI) assay (WHO 2002). -14 and modified Vero strain antibody titer.

The antigenic performance of NIBRG-14, Vero-15 and Vero-16 was determined by HI titration, and the results are shown in Table 2. The results indicated that the three viruses had similar antigenicity. Both Vero-15 and Vero-16 are suitable for use in vaccine production.

I would like to thank all those who are good at changing the above embodiments without departing from these broad and thorough concepts. Therefore, it is to be understood that the invention is not limited the

The above summary, as well as a detailed description of the preferred embodiments of the invention, will be readily understood as To illustrate the invention, the following chart showing the presently preferred embodiment. However, what should be known is this issue. The description is not limited by the particular embodiment shown.

In the chart.

Fig. 1 is a view showing the plaque morphology of the virus strain NIBRG-14 (Fig. 1A), influenza virus strain vero-15 (Fig. 1B) and vero-16 (Fig. 1C) after infection of African green monkey kidney cells. (Fig. 1A) 6 days after NIBRG-14 infection of African green monkey kidney cells. (Fig. 1B) and (Fig. 1C) are the plaque patterns of influenza virus strain vero-15 and vero-16 infected with African green monkey kidney cells for 3 days, respectively.

Figure 2 illustrates the viral titer of NIBRG-14 in different infection density (MOI) infections of African green monkey kidney cells at different Days Post Infection (DPI), titrated with HA (50ul), respectively (2A Figure) or plaque assay (PFU/ml) (Figure 2B) for titer detection.

Figure 3 is a diagram showing the viral titer of vero-15 infected with African green monkey kidney cells at different infection densities (MOI) at different days of infection (DPI), titrated with HA (50 ul) (Fig. 3A) or bacteria Spot analysis (PFU/ml) (Fig. 3B) for potency detection.

Figure 4 shows the viral titer of vero-16 infected with African green monkey kidney cells at different infection densities (MOI) at different days of infection (DPI), titrated with HA (50 ul) (Fig. 4A) or bacteria Spot analysis (PFU/ml) (Fig. 4B) for potency detection.

Figure 5 is a diagram showing the amino acid sequence (Fig. 5B) of the influenza virus polymerase protein (PB1) of vero-15, vero-16 and NIBRG-14, and their corresponding nucleotides (NT) ) Sequence (Figure 5A).

Figure 6 is a diagram showing the sequence alignment of the influenza virus polymerase protein (PB2) of vero-15, vero-16 and NIBRG-14, the amino acid sequence (Fig. 6B) and its corresponding nucleotide (NT) ) Sequence (Figure 6A).

Figure 7 is a diagram showing the sequence alignment of the influenza virus nucleoprotein (NP) of vero-15, vero-16 and NIBRG-14, the amino acid sequence (Fig. 7B) and its corresponding nucleotide (NT) Sequence (Figure 7A).

Figure 8 shows the sequence alignment analysis vero-15, vero-16 and NIBRG-14 influenza virus interstitial protein (M), amino acid sequence (Fig. 8B) and its corresponding nucleotide (NT) sequence (Fig. 8A).

Figure 9 is a diagram showing the sequence alignment of the influenza virus polymerase protein (PA) of vero-15, vero-16 and NIBRG-14, the amino acid sequence (Fig. 9B) and its corresponding nucleotide (NT) ) Sequence (Figure 9A).

Figure 10 is a diagram showing the amino acid sequence of the influenza virus non-structural protein (NS) of vero-15, vero-16 and NIBRG-14 (Fig. 10B) and its corresponding nucleotide (NT) ) Sequence (Figure 10A).

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Claims (17)

An isolated domesticated influenza A virus strain comprising: (a) a modified influenza polymerase protein (PB1), which is substituted with a white amine at position 195 of the PBR protein of the NIBRG-14 strain (SEQ ID NO: 1) (b) a modified influenza polymerase protein (PB2), which is substituted with a tyrosine group at position 359 of the PB2 protein (SEQ ID NO: 3) of the NIBRG-14 strain; (c) a modified influenza Polymerase protein (PA), which replaces the 465 position of the PA protein of the NIBRG-14 strain (SEQ ID NO: 9) with a histidine group, or the 494 position with a glycine group; and (d) Modification of influenza non-structural proteins (NS), including (i) substitution of the proline group at positions 90 and 110 of the PB1 protein (SEQ ID NO: 11) of the NIBRG-14 strain; or (ii) NIBRG-14 The 122 strain of the PB1 protein (SEQ ID NO: 11) of the virus strain was substituted with a stop codon; wherein, compared with the NIBRG-14 virus strain, the production of the acclimated influenza A virus strain in African green monkey kidney cells was increased. The influenza virus strain according to the first aspect of the patent application further includes: a modified influenza nucleoprotein (NP), which is substituted with an aspartic acid at position 255 of the NP protein (SEQ ID NO: 5) of the NIBRG-14 virus strain. The acid group, and at positions 410 and 446, are substituted with amphetamine groups. According to the influenza virus strain of the first aspect of the patent application, the method further comprises: a modified influenza interstitial protein (M), which is substituted with phenylalanine at position 236 of the M protein of the NIBRG-14 strain (SEQ ID NO: 7). base. The influenza virus strain according to item 1 of the patent application scope further includes influenza hemagglutinin protein (HA) (SEQ ID NO: 13) and influenza nucleoprotein (SEQ ID NO: 15) identical to the NIBRG-14 virus strain. According to the influenza virus strain of the first aspect of the patent application, the gene family includes: (a) a modified influenza PB1 gene, which is a PB1 group of the NIBRG-14 virus strain. 585, 584 and 1257 of the nucleotide sequence (SEQ ID NO: 2) were substituted with three thymines, respectively, and substituted at 1737 with adenine; (b) a modified influenza PB2 gene, which was NIBRG-14 The nucleotide sequence of the PB2 gene (SEQ ID NO: 4) of the virus strain was substituted with adenine; (c) a modified influenza PA gene, which is the nucleotide sequence of the PA gene of the NIBRG-14 strain (sequence recognition) No.: 1) is substituted for guanine, or substituted with cytosine at 895, 1054 and 1394, respectively; and (d) a modified influenza NS gene, which is the NS gene nucleoside of NIBRG-14 strain. 271, 331 and 335 of the acid sequence (SEQ ID NO: 12) were substituted with three cytosines, respectively, or deleted at 365. The influenza virus strain according to item 5 of the patent application scope further includes: a modified influenza NP gene, wherein 763 of the nucleotide sequence of the NP gene of the NIBRG-14 virus strain (SEQ ID NO: 6) is substituted with guanine, And at 921, 1229, 1337 and 1413, respectively, replaced by four thymines. The domesticated influenza virus strain according to item 5 of the patent application scope further includes: a modified influenza M gene, wherein the nucleotide sequence of the M gene of the NIBRG-14 virus strain (SEQ ID NO: 8) is replaced by 694 and 706, respectively. For two thymines. The influenza virus strain according to item 5 of the patent application scope further includes: (a) a nucleotide sequence of the influenza HA gene identical to the NIBRG-14 virus strain (SEQ ID NO: 14); and (b) one with NIBRG-14 The viral strain has the same influenza NA gene nucleotide sequence (SEQ ID NO: 16). An isolated domesticated influenza A virus strain comprising: (a) a modified influenza polymerase protein (PB1), which replaces 195 of the PBR protein of the NIBRG-14 strain (SEQ ID NO: 1) with leucine (b) a modified influenza polymerase protein (PB2), which is substituted with 359 of the PBR2 protein of the NIBRG-14 strain (SEQ ID NO: 3) as tyrosine; (c) a modified influenza polymerase protein ( PA), which replaces 494 of the PA protein (SEQ ID NO: 9) of the NIBRG-14 virus strain with glycine; (d) A modified influenza non-structural protein (NS) in which the 90 and 110 of the NS protein (SEQ ID NO: 11) of the NIBRG-14 strain are substituted with proline. The influenza virus strain according to item 9 of the patent application scope further includes an amino acid sequence (SEQ ID NO: 5) of influenza nucleoprotein (NP) identical to the NIBRG-14 virus strain, and an amine of influenza interstitial protein (M). Amino acid sequence (SEQ ID NO: 7), amino acid sequence of influenza hemagglutinin protein (HA) (SEQ ID NO: 13), and amino acid sequence of influenza neuraminidase protein (NA) (sequence recognition) No.: 15). According to the influenza virus strain of claim 10, the genetic composition includes the following nucleotide sequences: the same NP gene (SEQ ID NO: 6) and M gene (SEQ ID NO: 8), which are the same as the NIBRG-14 virus strain, HA gene (SEQ ID NO: 14), and NA gene (SEQ ID NO: 16); PBR1 gene of NIBRG-14 strain (SEQ ID NO: 2), substituted with thymine at 583, 584, and 1257, respectively. And substituted at 1737 with adenine; the PBR2 gene of NIBRG-14 strain (SEQ ID NO: 4), substituted for adenine at 1077; PA gene of NIBRG-14 strain (SEQ ID NO: 10), The 1482 was substituted with guanine; and the NS gene of the NIBRG-14 strain (SEQ ID NO: 12) was substituted with cytosine at 271, 331 and 335, respectively. An isolated domesticated influenza A virus strain comprising: (a) a modified influenza polymerase protein (PB1), which replaces 195 of the PBR protein of the NIBRG-14 strain (SEQ ID NO: 1) with leucine (b) a modified influenza polymerase protein (PB2), which replaces 359 of the PB2 protein of NIBRG-14 strain (SEQ ID NO: 3) with tyrosine; (c) a modified influenza nucleoprotein (NP) ), 255 of the NP protein (SEQ ID NO: 5) of the NIBRG-14 strain was substituted with aspartic acid, and 410 and 446 were substituted with phenylalanine; (d) A modified influenza interstitial protein (M), which replaces 236 of the M protein of the NIBRG-14 strain (SEQ ID NO: 7) with phenylalanine; (e) a modified influenza polymerase protein (PA) , replacing 494 of the PA protein (SEQ ID NO: 9) of the NIBRG-14 strain with histidine; and (f) a modified influenza non-structural protein (NS), which is the NS of the NIBRG-14 strain. The 122 position of the protein (sequence number: 11) was replaced with a stop code. The influenza virus strain according to item 12 of the patent application scope further includes an amino acid sequence (SEQ ID NO: 13) of influenza hemagglutinin protein (HA) which is identical to the NIBRG-14 virus strain, and influenza neuraminidase protein ( NA) amino acid sequence (SEQ ID NO: 15). According to the influenza virus strain of the 13th patent application, the genetic composition includes the following nucleotide sequences: the same HA gene (SEQ ID NO: 14) and NA gene (SEQ ID NO: 16) as the NIBRG-14 strain; The PB1 gene of NIBRG-14 strain (SEQ ID NO: 2) was substituted with thymine at 583, 584 and 1257, and adenine at 1737; PB2 gene of NIBRG-14 strain (sequence identification number) :4), substituted at 1077 for adenine; NP gene of NIBRG-14 strain (SEQ ID NO: 6), substituted for guanine at 763, and replaced with thymus at 921, 1229, 1337 and 1413, respectively Pyrimidine; the M gene of the NIBRG-14 strain (SEQ ID NO: 8), substituted with thymine at 694 and 706, respectively; the PA gene of NIBRG-14 strain (SEQ ID NO: 10), at 895, 1054 and 1394 was replaced with three cytosines; and the NS gene of NIBRG-14 strain (SEQ ID NO: 12), and nucleotides were deleted at 365. An influenza vaccine comprising a domesticated influenza A virus strain according to item 1 of the patent application and a pharmaceutically acceptable carrier. An influenza vaccine comprising an acclimated influenza A virus strain according to item 9 of the patent application and a pharmaceutically acceptable carrier. An influenza vaccine comprising a domesticated influenza A virus strain according to item 12 of the patent application and a pharmaceutically acceptable carrier.